ABSTRACT Recent studies have demonstrated that post-transcriptional mRNA modifications can regulate gene expression, with implications for cellular differentiation, development, and cancer. For example, the m6A modification can regulate fundamental aspects of mRNA biology, such as splicing, stability, and translation. In contrast, much less is known about internal 2'-O-methylation (Nm) on mRNA. Now, using genetic models, our data suggest that small nucleolar RNAs (snoRNAs) can guide internal Nm modification of mRNA, via the enzyme fibrillarin (FBL). Our findings also suggest that Nm modification can regulate mRNA levels and protein expression in vivo. In this application, we propose to define the mechanisms that lead to Nm modification of mRNA, and to understand how Nm regulates gene expression. Our long-term goals are to determine how snoRNA-guided modifications of mRNA impact gene expression, cell biology, and organism health. The objective of this proposal is to specifically determine how snoRNA-guided Nm modification regulates the expression of Pxdn mRNA. Our central hypothesis is that Rpl13a snoRNAs guide FBL-mediated Nm modification of Pxdn mRNA, and that the modification increases mRNA abundance but inhibits protein translation. We propose to test our hypothesis with the following Specific Aims: (1) Define the snoRNA(s) and protein components that target Pxdn mRNA for Nm modification; (2) Define how snoRNA-guided modification of Pxdn regulates RNA and protein expression; and (3) Discover the network of snoRNA-guided Nm modifications in cells. Successful completion of these Aims will define, for the first time, how Nm modification of mRNA regulates gene expression. We also expect that our work will define a new role for snoRNAs in the modification of mRNA. As such, this work will open new avenues in the study of both RNA modifications and snoRNA biology.